Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and documents is incorporated by reference in its entirety herein.
A number of techniques have been developed that facilitate rapid and accurate detection of infectious agents, such as viruses, bacteria and fungi. These methods may also be applied to the detection and differentiation of normal and abnormal genes. Most of these protocols employ exponential amplification of minute amounts of a target nucleic acid sequence (e.g., DNA or RNA) in a test sample. These include the polymerase chain reaction (PCR) (Saiki et al., Science 230:1350, 1985; Saiki et al., Science 239:487, 1988; PCR Technology, Henry A. Erlich, ed., Stockton Press, 1989; Patterson et al., Science 260:976, 1993), ligase chain reaction (LCR) (Barany, Proc. Natl. Acad. Sci. USA 88:189, 1991), strand displacement amplification (SDA) (Walker et al., Nucl. Acids Res. 20:1691, 1992), Qβ replicase amplification (QβRA) (Wu et al., Proc. Natl. Acad. Sci. USA 89:11769, 1992; Lomeli et al., Clin. Chem. 35:1826, 1989), nucleic acid sequence based amplification (NASBA), and self-sustained replication (3SR) (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990). While all of these techniques are powerful tools for the detection and identification of trace amounts of a target nucleic acid in a sample, they all exhibit various shortcomings, which have prevented their general applicability as routine diagnostic techniques in clinical laboratory settings.
The preparation of the target nucleic acid, for example, is a procedural impediment required for subsequent steps such as amplification and detection. Target nucleic acid preparation is time and labor intensive and, thus, generally unsuitable for a clinical setting, where rapid and accurate results are required. Another problem, which is particularly pronounced when using PCR and SDA, is the necessity for empirically determining optimal conditions for target nucleic acid amplification for each target. Moreover, conditions required for standardizing quantitation assessments can also vary from sample to sample. This lack of precision manifests itself most dramatically when the diagnostic assay is implemented in multiplex format, that is, in a format designed for the simultaneous detection of several different target sequences.
Thus, the development of more rapid and less technically challenging protocols for detecting trace amounts of nucleic acid sequences associated with or indicative of the presence of a pathogen, for example, would be useful for clinical diagnostic screening assays.